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Abstract:

Compositions and methods for the diagnosis and prevention of B. abortus
infection are provided.

Claims:

1. A method of detecting a Brucella abortus infection in an animal, said
method comprising: a) obtaining a biological sample from said animal; and
b) detecting the presence of at least one antibody immunologically
specific for at least one Brucella abortus protein selected from Table 1,
wherein the presence of antibodies to the Brucella abortus protein
indicates a Brucella abortus infection in said animal.

2. The method of claim 1, wherein at least one of said Brucella abortus
protein is selected from the group consisting of malate dehydrogenase,
D15, and AfuA.

3. The method of claim 2, wherein at least one of said Brucella abortus
protein is malate dehydrogenase or D15.

4. The method of claim 3, wherein at least one of said Brucella abortus
protein is malate dehydrogenase.

5. The method of claim 2, wherein said malate dehydrogenase comprises an
amino acid sequence having at least 95% homology with SEQ ID NO: 2; said
D15 comprises an amino acid sequence having at least 95% homology with
SEQ ID NO: 1; and said AfuA comprises an amino acid sequence having at
least 95% homology with SEQ ID NO: 3.

6. The method of claim 1, wherein said biological sample is blood or
serum.

7. A method of inhibiting a Brucella abortus infection in an animal, said
method comprising administering a composition to said animal wherein said
composition comprises at least one Brucella abortus protein selected from
Table 1 and at least one pharmaceutically acceptable carrier.

8. The method of claim 7, wherein at least one of said Brucella abortus
protein is selected from the group consisting of malate dehydrogenase,
D15, and AfuA.

9. The method of claim 8, wherein at least one of said Brucella abortus
protein is malate dehydrogenase or D15.

10. The method of claim 9, wherein at least one of said Brucella abortus
protein is malate dehydrogenase.

11. The method of claim 8, wherein said malate dehydrogenase comprises an
amino acid sequence having at least 95% homology with SEQ ID NO: 2; said
D15 comprises an amino acid sequence having at least 95% homology with
SEQ ID NO: 1; and said AfuA comprises an amino acid sequence having at
least 95% homology with SEQ ID NO: 3.

12. The method of claim 7, further comprising the administration of at
least one other Brucella abortus vaccine.

13. A composition comprising at least one isolated Brucella abortus
protein selected from Table 1 and at least one pharmaceutically
acceptable carrier.

14. The composition of claim 13, wherein at least one of said Brucella
abortus protein is selected from the group consisting of malate
dehydrogenase, D15, and AfuA.

15. The composition of claim 13, wherein said composition comprises at
least two Brucella abortus proteins selected from the group consisting of
malate dehydrogenase, D15, and AfuA.

[0002] The present invention relates to the fields of diagnosing and
preventing microbial infections. More specifically, the invention
provides compositions for the detection of Brucella abortus and
compositions to inhibit/vaccinate against Brucella abortus infections.

BACKGROUND OF THE INVENTION

[0003] The threat of brucellosis is of particular concern because of its
potential to disrupt agricultural economy, and the disease continues to
be problematic in parts of the U.S. today. To compound the problem, elk
and wild bison within the Greater Yellowstone Area (GYA) are major
reservoirs for brucellosis and cattle may contract Brucella abortus from
this population (Godfroid, J. (2002) Rev. Sci. Technol. Off. Int. Epiz.,
21:277-286). Wildlife Brucella reservoirs represent a major obstacle to
the development of an effective eradication program focusing on domestic
livestock in Wyoming. Therefore, the Wyoming Brucellosis Coordination
Team has issued a series of management practices aimed at controlling
brucellosis in elk. Among these practices is a recommendation to test
feed ground elk as a way to monitor sero-prevalence and efficacy of
brucellosis elimination activities.

[0004] Unfortunately, diagnostic methods for brucellosis have been limited
because of the lack of consistently reliable targets which ensure high
specificity and sensitivity. More sensitive than traditional Brucella
diagnostic methods, serologic diagnosis based on reactivity to LPS has
been reported (Saegerman et al. (2004) Vet. Microbiol., 100:91-105).
Geographic areas of false positive serologic reactions exist however,
which reduce specificity of such assays (Saegerman et al. (2004) Vet.
Microbial., 100:91-105). More recently, PCR-based tests have been
evaluated as a next-generation approach to early diagnosis/detection,
although standardization of methodologies and a more diverse repertoire
of target genes still need to be established (Al Dahouk et al. (2004)
Clin. Lab., 50:387-394; Navarro et al. (2004) Exp. Rev. Mol. Diag.,
4:115-123).

[0005] While several genes and their products associated with Brucella
virulence have been described (for review, see Ko et al. (2003) Clin.
Microbiol. Rev., 16:65-78), most have been identified using in
vitro-grown bacteria. In this approach, host factors important in
up-regulating some virulence loci may not be present in laboratory-grown
cultures. Signature Tagged Mutagenesis (STM) has been employed with
Brucella spp. in an attempt to identify virulence genes which are
requisite to survival in vivo (Hong et al. (2000) Infect. Immun.,
68:4102-4107; Zygmunt et al. (2006) Microb. Infect., 8:2849-2854). This
technique involves a "negative" selection approach which relies on live
animals to only identify mutations in those genes essential for host
survival, and not immunogenic gene products. The method is also quite
sensitive to experimental variables and to date has yielded limited
information on molecular aspects of Brucella virulence. A less cumbersome
and less artifactual approach is to utilize immune sera adsorbed with the
in vitro-grown pathogen as a screening reagent for those gene products
relevant to in vivo survival and pathogenesis. This technique is known as
In vivo-Induced Antigen Technology, (IVIAT), and has been successfully
used on bacterial pathogens to identify antigenic proteins expressed
during infection (Handfield et al. (2000) Trends Microbiol., 8:336-339;
Rollins et al. (2005) Cell. Microbiol., 7:1-9). Most recently, IVIAT has
been applied to Bacillus anthracis to identify potential diagnostic,
vaccine, and therapeutic candidates (Rollins et al. (2008) PLoS One
3:e1824). IVIAT has also been used with other facultative intracellular
pathogens, such as Mycobacterium tuberculosis (Deb et al. (2002)
Tuberculosis 82:175-182) and Legionella pneumophila (Chang et al. (2005)
Infect. Immun., 73:4272-4280).

SUMMARY OF THE INVENTION

[0006] In accordance with the instant invention, methods of detecting a
Brucella infection, particularly a Brucella abortus infection, in an
animal are provided. In a particular embodiment, the method comprises a)
obtaining a biological sample from the animal; and b) detecting the
presence of at least one antibody immunologically specific for at least
one Brucella abortus protein, wherein the presence of antibodies to the
Brucella abortus protein indicates a Brucella abortus infection in the
animal. In another embodiment, at least one Brucella abortus protein is
selected from the examples hereinbelow, particularly Table 1, more
particularly at least one Brucella abortus protein is malate
dehydrogenase (Mdh), D15, or AfuA. In one embodiment, the methods allow
for the differentiation between naturally infected and immunized animals
and/or the differentiation based on B. abortus strains. In still another
embodiment, compositions and kits are provided for the practice of the
detection methods.

[0007] According to another aspect of the instant invention, methods of
inhibiting a Brucella infection, particularly a Brucella abortus
infection, in an animal are provided. In a particular embodiment, the
method comprises administering to an animal at least one composition
comprising at least one Brucella abortus protein, particularly one
selected from the examples hereinbelow (particularly Table 1), and at
least one pharmaceutically acceptable carrier. In one embodiment, at
least one of the Brucella abortus proteins is malate dehydrogenase (Mdh),
D15, or AfuA. In yet another embodiment, the method further comprises the
administration of at least one other Brucella abortus vaccine and/or
anti-microbial agent.

[0008] In accordance with yet another aspect, compositions comprising at
least one Brucella abortus protein, particularly one selected from the
examples hereinbelow (particularly Table 1), and at least one
pharmaceutically acceptable carrier are provided. The compositions may be
used to inhibit, treat, or prevent a Brucella abortus infection, e.g., as
a vaccine against a Brucella abortus infection.

BRIEF DESCRIPTIONS OF THE DRAWING

[0009] FIG. 1 provides representative Western blots of recombinant AfuA,
Mdh, and D15 against positive and negative serum samples. Molecular
weights of the recombinant proteins are indicated in parentheses.

[0017]FIG. 9 provides a graph of IL-10 secretion from mouse macrophages
exposed to various agents.

[0018]FIG. 10 provides a graph showing IVI genes upregulated in vivo
during S19 infection. Average fold change of bacterial mRNA isolated from
five mice infected with S19 at each time point compared to in vitro-grown
B. abortus S19.

[0020] IVIAT is used herein to identify bacterial antigens relevant to the
survival of B. abortus in elk and other mammals, with the anticipated
outcome of determining what virulence effectors are important in this
host-pathogen system, as well as to identify new diagnostic targets
and/or sub-unit vaccine candidates that can be applied to different
susceptible hosts.

[0021] Elk in the Greater Yellowstone Area are a major reservoir for
brucellosis, which represents an obstacle to eradication of the disease
in domestic livestock. Furthermore, immune responses to Brucella abortus
infection in the wild host are not well-understood. In this regard, in
vivo-induced antigen technology (IVIAT) was employed to identify novel B.
abortus antigens expressed during infection in elk. Sera collected from
sero-positive Wyoming elk were pooled and absorbed against in vitro-grown
cultures of B. abortus. Approximately 35,000 E. coli clones, expressing
B. abortus DNA, were then screened by colony immunoblot, yielding ten
genes with immuno-reactive products, to include seven proteins secreted
beyond the inner membrane. Three products--an outer membrane protein
(D15), malate dehydrogenase (Mdh), and an ion transporter (AfuA)--were
examined by Western blot against individual elk serum samples.
Sero-reactivity was significantly more frequent for both Mdh and D15 in
naturally infected animals, compared to vaccinated and uninfected elk,
indicating that antibody to these two antigens is a predictor of natural
infection. Cross-reactivity of all three proteins was next examined with
serum samples from confirmed brucellosis-positive cattle. While variable
patterns of reactivity were seen with the antigens, the sample group was
equivalently reactive to AfuA and Mdh, compared to elk, indicating that
these antigens are commonly expressed during infection in both hosts.
Therefore, the application of IVIAT to B. abortus not only facilitates
the identification of serologic markers for brucellosis in elk, but
provides further insight into biological processes of the pathogen in
different hosts.

[0022] With the application of the IVIAT gene discovery methodology to B.
abortus, ten genes were identified which are expressed during infection
and whose products are recognized by the cervid immune system. This
outcome was the result of screening approximately 35,000 E. coli clones
containing Brucella DNA with elk serum extensively adsorbed with in
vitro-grown bacteria to remove antibodies to constitutively expressed
proteins. Previous reports support the hypothesis that some of the genes
are expressed during Brucella infection in several different hosts
(Chirhart-Gilleland et al. (1998) Infect. Immun., 66:4000-4003; Ko et al.
(2003) Clin. Microbiol. Rev., 16:65-78; Caro-Hernandez et al. (2007)
Infect. Immun., 75:4050-4061), and that the application of IVIAT has
further defined these antigens as being up-regulated in vivo. Unlike
previous genes identified by STM (Hong et al. (2000) Infect. Immun.,
68:4102-4107; Zygmunt et al. (2006) Microb. Infect., 8:2849-2854.), at
least 30% of the loci identified through IVIAT are predicted to encode
outer membrane proteins, which intuitively would be the candidates of
choice for further examination as to their role in Brucella virulence.
With this intent, the remaining antigens in this category, as well as the
identified periplasmic proteins, can also be further characterized since
their relevance to the survival of this microorganism in vivo cannot be
discounted (Tang et al. (2005) J. Bacteriol., 187:6231-6237; Miranda et
al. (2004) Infect. Immun., 72:1666-1676; Anderson et al. (2009) Infect
Immun., 77:3466-74); Vines et al. (2005) J. Bacteriol., 187:3359-3368;
Comerci et al. (2001) Cell Microbiol., 3:159-168; Roux et al. (2007) Cell
Microbiol., 9:1851-1869).

[0023] In addition to detecting novel virulence genes, IVIAT has provided
the means to identify B. abortus antigenic gene products as markers for
infection. From the set of three recombinant IVIAT-identified proteins
that were expressed and examined for sero-prevalence, a pattern of
reactivity has emerged from serum collected from Wyoming elk. Antibody
reactivity to two of the selected antigens, Mdh and D15, was shown to be
a predictor of natural infection in this host. Moreover, an equivalent
frequency of reactivity of at least one gene product, Mdh, was seen in
immune domestic livestock, indicating that common biologic processes
associated with this enzyme are utilized by B. abortus in different
hosts. Accordingly, based on the observations in this study, the utility
of these antigens extends to the diagnosis of brucellosis in other
animals, such as domestic animals.

[0024] Although certain laboratory-based assays are capable of
differentiating between B. abortus S19-vaccinated elk, and naturally
infected animals (Van Houten et al. (2003) J. Wildl. Dis., 39:316-322.;
Gall et al. (2001) J. Wildl. Dis., 37:110-118), both methods require
operator training, are fairly labor intensive, and are not easily
amendable to field application. The results provided herein demonstrate
the potential for immobilized IVIAT-identified antigens to differentiate
between vaccinated and naturally infected animals. This type of immune
"footprinting" can also be useful for such differentiation in other
susceptible hosts, and provide the basis of a new field deployable, rapid
assay for the diagnosis of brucellosis in elk and/or domestic livestock.
Such an assay, e.g., in a lateral flow device platform, has been
developed for early detection and monitoring of other bacterial pathogens
(Biagini et al. (2006) Clin. Vac. Immunol., 13:541-546). Furthermore, the
cloning, expression and evaluation of additional in vivo-expressed genes
may reveal additional patterns of humoral immunity which differentiate
between infection by different B. abortus strains. Indeed, the IVIAT
method can be repeated on other strains to identify further strain
specific antigens.

[0025] Although B. abortus genes and their products identified through
IVIAT can be further evaluated as to their exact role(s) in virulence,
this approach can be used to identify antigens which are useful in
generating protective immune responses in multiple hosts. The finding
herein that two proteins were reactive with equivalent frequency in both
cervid and bovine hosts (AfuA and Mdh) supports this conclusion.

I. Definitions

[0026] "Nucleic acid" or a "nucleic acid molecule" as used herein refers
to any DNA or RNA molecule, either single or double stranded and, if
single stranded, the molecule of its complementary sequence in either
linear or circular form. In discussing nucleic acid molecules, a sequence
or structure of a particular nucleic acid molecule may be described
herein according to the normal convention of providing the sequence in
the 5' to 3' direction. With reference to nucleic acids of the invention,
the term "isolated nucleic acid" is sometimes used. This term, when
applied to DNA, refers to a DNA molecule that is separated from sequences
with which it is immediately contiguous in the naturally occurring genome
of the organism in which it originated. For example, an "isolated nucleic
acid" may comprise a DNA molecule inserted into a vector, such as a
plasmid or virus vector, or integrated into the genomic DNA of a
prokaryotic or eukaryotic cell or host organism.

[0027] When applied to RNA, the term "isolated nucleic acid" refers
primarily to an RNA molecule encoded by an isolated DNA molecule as
defined above. Alternatively, the term may refer to an RNA molecule that
has been sufficiently separated from other nucleic acids with which it
would be associated in its natural state (i.e., in cells or tissues). An
"isolated nucleic acid" (either DNA or RNA) may further represent a
molecule produced directly by biological or synthetic means and separated
from other components present during its production.

[0028] A "vector" is a replicon, such as a plasmid, cosmid, bacmid, phage
or virus, to which another genetic sequence or element (either DNA or
RNA) may be attached so as to bring about the replication of the attached
sequence or element.

[0029] An "expression operon" refers to a nucleic acid segment that may
possess transcriptional and translational control sequences, such as
promoters, enhancers, translational start signals (e.g., ATG or AUG
codons), polyadenylation signals, terminators, and the like, and which
facilitate the expression of a polypeptide coding sequence in a host cell
or organism.

[0030] The term "substantially pure" refers to a preparation comprising at
least 50-60% by weight of a given material (e.g., nucleic acid,
oligonucleotide, protein, etc.). More preferably, the preparation
comprises at least 75% by weight, and most preferably 90-95% by weight of
the given compound. Purity is measured by methods appropriate for the
given compound (e.g., chromatographic methods, agarose or polyacrylamide
gel electrophoresis, HPLC analysis, and the like).

[0031] The term "oligonucleotide" as used herein refers to sequences,
primers and probes of the present invention, and is defined as a nucleic
acid molecule comprised of two or more ribo- or deoxyribonucleotides,
preferably more than three. The exact size of the oligonucleotide will
depend on various factors and on the particular application and use of
the oligonucleotide.

[0032] The term "primer" as used herein refers to an oligonucleotide,
either RNA or DNA, either single-stranded or double-stranded, either
derived from a biological system, generated by restriction enzyme
digestion, or produced synthetically which, when placed in the proper
environment, is able to functionally act as an initiator of
template-dependent nucleic acid synthesis. When presented with an
appropriate nucleic acid template, suitable nucleoside triphosphate
precursors of nucleic acids, a polymerase enzyme, suitable cofactors and
conditions such as appropriate temperature and pH, the primer may be
extended at its 3' terminus by the addition of nucleotides by the action
of a polymerase or similar activity to yield a primer extension product.
The primer may vary in length depending on the particular conditions and
requirement of the application. For example, in diagnostic applications,
the oligonucleotide primer is typically 15-25 or more nucleotides in
length. The primer must be of sufficient complementarity to the desired
template to prime the synthesis of the desired extension product, that
is, to be able to anneal with the desired template strand in a manner
sufficient to provide the 3' hydroxyl moiety of the primer in appropriate
juxtaposition for use in the initiation of synthesis by a polymerase or
similar enzyme. It is not required that the primer sequence represent an
exact complement of the desired template. For example, a
non-complementary nucleotide sequence may be attached to the 5' end of an
otherwise complementary primer. Alternatively, non-complementary bases
may be interspersed within the oligonucleotide primer sequence, provided
that the primer sequence has sufficient complementarity with the sequence
of the desired template strand to functionally provide a template-primer
complex for the synthesis of the extension product.

[0033] The term "probe" as used herein refers to an oligonucleotide,
polynucleotide or nucleic acid, either RNA or DNA, whether occurring
naturally as in a purified restriction enzyme digest or produced
synthetically, which is capable of annealing with or specifically
hybridizing to a nucleic acid with sequences complementary to the probe.
A probe may be either single-stranded or double-stranded. The exact
length of the probe will depend upon many factors, including temperature,
source of probe and use of the method. For example, for diagnostic
applications, depending on the complexity of the target sequence, the
oligonucleotide probe typically contains 15-25, 15-30, or more
nucleotides, although it may contain fewer nucleotides. The probes herein
are selected to be complementary to different strands of a particular
target nucleic acid sequence. This means that the probes must be
sufficiently complementary so as to be able to "specifically hybridize"
or anneal with their respective target strands under a set of
pre-determined conditions. Therefore, the probe sequence need not reflect
the exact complementary sequence of the target. For example, a
non-complementary nucleotide fragment may be attached to the 5' or 3' end
of the probe, with the remainder of the probe sequence being
complementary to the target strand. Alternatively, non-complementary
bases or longer sequences can be interspersed into the probe, provided
that the probe sequence has sufficient complementarity with the sequence
of the target nucleic acid to anneal therewith specifically.

[0034] Polymerase chain reaction (PCR) has been described in U.S. Pat.
Nos. 4,683,195, 4,800,195, and 4,965,188, the entire disclosures of which
are incorporated by reference herein.

[0035] With respect to single stranded nucleic acids, particularly
oligonucleotides, the term "specifically hybridizing" refers to the
association between two single-stranded nucleotide molecules of
sufficiently complementary sequence to permit such hybridization under
pre-determined conditions generally used in the art (sometimes termed
"substantially complementary"). In particular, the term refers to
hybridization of an oligonucleotide with a substantially complementary
sequence contained within a single-stranded DNA molecule of the
invention, to the substantial exclusion of hybridization of the
oligonucleotide with single-stranded nucleic acids of non-complementary
sequence. Appropriate conditions enabling specific hybridization of
single stranded nucleic acid molecules of varying complementarity are
well known in the art.

[0037] As an illustration of the above formula, using [Na+]=[0.368] and
50% formamide, with GC content of 42% and an average probe size of 200
bases, the Tm is 57° C. The Tm of a DNA duplex decreases
by 1-1.5° C. with every 1% decrease in homology. Thus, targets
with greater than about 75% sequence identity would be observed using a
hybridization temperature of 42° C.

[0038] The stringency of the hybridization and wash depend primarily on
the salt concentration and temperature of the solutions. In general, to
maximize the rate of annealing of the probe with its target, the
hybridization is usually carried out at salt and temperature conditions
that are 20-25° C. below the calculated Tm of the hybrid.
Wash conditions should be as stringent as possible for the degree of
identity of the probe for the target. In general, wash conditions are
selected to be approximately 12-20° C. below the Tm of the
hybrid. In regards to the nucleic acids of the current invention, a
moderate stringency hybridization is defined as hybridization in
6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml
denatured salmon sperm DNA at 42° C., and washed in 2×SSC
and 0.5% SDS at 55° C. for 15 minutes. A high stringency
hybridization is defined as hybridization in 6×SSC,
5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon
sperm DNA at 42° C., and washed in 1×SSC and 0.5% SDS at
65° C. for 15 minutes. A very high stringency hybridization is
defined as hybridization in 6×SSC, 5×Denhardt's solution,
0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C.,
and washed in 0.1×SSC and 0.5% SDS at 65° C. for 15 minutes.

[0039] The term "isolated protein" or "isolated and purified protein" is
sometimes used herein. This term refers primarily to a protein produced
by expression of an isolated nucleic acid molecule of the invention.
Alternatively, this term may refer to a protein that has been
sufficiently separated from other proteins with which it would naturally
be associated, so as to exist in "substantially pure" form. "Isolated" is
not meant to exclude artificial or synthetic mixtures with other
compounds or materials, or the presence of impurities that do not
interfere with the fundamental activity, and that may be present, for
example, due to incomplete purification, or the addition of stabilizers.

[0040] The term "substantially pure" refers to a preparation comprising at
least 50-60% by weight of a given material (e.g., nucleic acid,
oligonucleotide, protein, etc.). More preferably, the preparation
comprises at least 75% by weight, and most preferably 90-95% or more by
weight of the given compound. Purity is measured by methods appropriate
for the given compound (e.g. chromatographic methods, agarose or
polyacrylamide gel electrophoresis, HPLC analysis, and the like).

[0041] The term "gene" refers to a nucleic acid comprising an open reading
frame encoding a polypeptide, including both exon and (optionally) intron
sequences. The nucleic acid may also optionally include non-coding
sequences such as promoter or enhancer sequences. The term "intron"
refers to a DNA sequence present in a given gene that is not translated
into protein and is generally found between exons.

[0042] "Pharmaceutically acceptable" indicates approval by a regulatory
agency of the Federal or a state government or listed in the U.S.
Pharmacopeia or other generally recognized pharmacopeia for use in
animals, and more particularly in humans.

[0044] A "therapeutically effective amount" of a compound or a
pharmaceutical composition refers to an amount effective to prevent,
inhibit, treat, or lessen the symptoms of a particular disorder or
disease. The treatment of a microbial infection (e.g., B. abortus
infection) herein may refer to curing, relieving, and/or preventing the
microbial infection, the symptom of it, or the predisposition towards it.

[0045] An "antibody" or "antibody molecule" is any immunoglobulin,
including antibodies and fragments thereof, that binds to a specific
antigen. The term includes polyclonal, monoclonal, chimeric, single
domain (Dab) and bispecific antibodies. As used herein, antibody or
antibody molecule contemplates recombinantly generated intact
immunoglobulin molecules and immunologically active portions of an
immunoglobulin molecule such as, without limitation: Fab, Fab',
F(ab')2, F(v), scFv, scFv2, scFv-Fc, minibody, diabody,
tetrabody, single variable domain (e.g., variable heavy domain, variable
light domain), and bispecific. Dabs can be composed of a single variable
light or heavy chain domain. The instant invention also encompasses
antibody mimetics such as Affibody® molecules (Affibody, Bromma,
Sweden) and peptabodies (Terskikh et al. (1997) PNAS 94:1663-1668).
Methods for producing antibodies (e.g., recombinantly) are well-known in
the art.

[0046] With respect to antibodies, the term "immunologically specific"
refers to antibodies that bind to one or more epitopes of a protein or
compound of interest, but which do not substantially recognize and bind
other molecules in a sample containing a mixed population of antigenic
biological molecules. The term "specifically binds" refers to the binding
of a polypeptide or compound of interest to a target polypeptide or
compound while not substantially recognizing and binding other molecules
in a sample containing a mixed population of biological molecules.

[0047] The phrases "affinity tag," "purification tag," and "epitope tag"
may all refer to tags that can be used to effect the purification of a
protein of interest. Purification/affinity/epitope tags are well known in
the art (see Sambrook et al., 2001, Molecular Cloning, Cold Spring Harbor
Laboratory) and include, but are not limited to: polyhistidine tags (e.g.
6×His), polyarginine tags, glutathione-S-transferase (GST), maltose
binding protein (MBP), S-tag, influenza virus HA tag, thioredoxin,
staphylococcal protein A tag, the FLAG® epitope, AviTag® epitope
(for subsequent biotinylation), dihydrofolate reductase (DHFR), an
antibody epitope (e.g., a sequence of amino acids recognized and bound by
an antibody), the c-myc epitope, and heme binding peptides.

[0049] As used herein, a "biological sample" refers to a sample of
biological material obtained from a subject including a tissue, a tissue
sample, a cell sample, and a biological fluid (e.g., blood, serum, or
urine). Preferably, the biological sample is blood or serum.

II. Polypeptides

[0050] The B. abortus proteins of the present invention may be prepared in
a variety of ways, according to known methods. In one embodiment, the B.
aborted proteins are produced recombinantly. The B. abortus proteins may
be purified from appropriate sources, e.g., bacterial or animal cultured
cells or tissues, optionally transformed, by immunoaffinity purification.
The availability of nucleic acid molecules encoding the B. abortus
proteins also enables production of the protein using in vitro expression
methods and cell-free expression systems known in the art. In vitro
transcription and translation systems are commercially available, e.g.,
from Promega Biotech (Madison, Wis.) or Gibco-BRL (Gaithersburg, Md.).

[0051] Larger quantities of B. abortus proteins may be produced by
expression in a suitable prokaryotic or eukaryotic system. For example,
part or all of a DNA molecule encoding for a B. abortus proteins may be
inserted into a plasmid vector adapted for expression in a bacterial
cell, such as E. coli. Such vectors comprise the regulatory elements
necessary for expression of the DNA in the host cell positioned in such a
manner as to permit expression of the DNA in the host cell. Such
regulatory elements required for expression include promoter sequences,
transcription initiation sequences and, optionally, enhancer sequences.

[0052] B. abortus proteins produced by gene expression in a recombinant
prokaryotic or eukaryotic system may be purified according to methods
known in the art. A commercially available expression/secretion system
can be used, whereby the recombinant protein is expressed and thereafter
secreted from the host cell, and readily purified from the surrounding
medium. If expression/secretion vectors are not used, an alternative
approach involves purifying the recombinant protein by affinity
separation, such as by immunological interaction with antibodies that
bind specifically to the recombinant protein or nickel columns for
isolation of recombinant proteins tagged with 6-8 histidine residues at
their N-terminus or C-terminus. The B. abortus proteins of the instant
invention may be linked to at least one purification tag, as described
herein. In a particular embodiment, the B. abortus protein is attached to
a 6×His tag. In still another embodiment, the B. abortus protein is
attached to the sequence
Met-Ala-His-His-His-His-His-His-Val-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 4) on
the amino terminus.

[0053] B. abortus proteins of the invention, prepared by the
aforementioned methods, may be analyzed according to standard procedures.
For example, such protein may be subjected to amino acid sequence
analysis, according to known methods.

[0054] B. abortus proteins of the instant invention are provided in the
examples hereinbelow, including those in Table 1 as well as PrpA, Hia,
and MltE (particularly Hia). In a particular embodiment, the B. abortus
proteins of the instant invention are selected from those provided in
Table 1. In yet another embodiment, the B. abortus proteins is selected
from the group consisting of D15, BA14K, Omp25d, malate dehydrogenase,
AfuA, TolA, and VirJ. The B. abortus proteins may also be selected from
the group consisting of Hia, D15, malate dehydrogenase, and AfuA. In
still another embodiment, the B. abortus proteins are selected from the
group consisting of D15, malate dehydrogenase, and AfuA. FIGS. 2-4
provide amino acid sequence of B. abortus D15, B. abortus malate
dehydrogenase, and B. abortus AfuA, respectively. The amino acid
sequences of the other proteins may be obtained through their GenBank ID
numbers (e.g., at www.ncbi.nlm.nih.gov/genbank/). The amino acid sequence
of the B. abortus proteins of the instant invention may have at least
75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% homology with the provided
sequence (e.g., SEQ ID NOs: 1-3 and 5 or those provided in GenBank),
particularly at least 90% or 95% homology.

[0055] The instant invention also encompasses antibodies immunologically
specific for the B. abortus proteins of the instant invention.

III. Nucleic Acid Molecules

[0056] Nucleic acid molecules encoding the B. abortus proteins of the
invention may be prepared by any method known in the art such as (1)
synthesis from appropriate nucleotide triphosphates or (2) isolation
and/or amplification from biological sources. The availability of
nucleotide sequence information enables preparation of an isolated
nucleic acid molecule of the invention by oligonucleotide synthesis.
Indeed, knowledge of the amino sequence is sufficient to determine an
encoding nucleic acid molecule. Synthetic oligonucleotides may be
prepared by the phosphoramidite method employed in the Applied Biosystems
38A DNA Synthesizer or similar devices. The resultant construct may be
purified according to methods known in the art, such as gel
electrophoresis or high performance liquid chromatography (HPLC).

[0057] Nucleic acid sequences encoding the B. abortus proteins of the
invention may be isolated from appropriate biological sources using
methods known in the art. In one embodiment, a cDNA clone of the B.
abortus proteins is isolated from a cDNA expression library and modified,
if necessary, to create the B. abortus proteins of the instant invention.
In an alternative embodiment, utilizing the sequence information provided
by the cDNA sequence, genomic clones encoding B. abortus proteins may be
isolated.

[0058] Nucleic acids of the present invention may be maintained in any
convenient vector, particularly an expression vector. Different promoters
may be utilized to drive expression of the nucleic acid sequences based
on the cell in which it is to be expressed. Antibiotic resistance markers
are also included in these vectors to enable selection of transformed
cells. B. abortus protein encoding nucleic acid molecules of the
invention include cDNA, genomic DNA, RNA, and fragments thereof which may
be single- or double-stranded. Thus, this invention provides
oligonucleotides having sequences capable of hybridizing with at least
one sequence of a nucleic acid molecule of the present invention.

[0059] Also encompassed in the scope of the present invention are
oligonucleotide probes which specifically hybridize with the B. abortus
protein nucleic acid molecules of the invention. Primers capable of
specifically amplifying B. abortus protein encoding nucleic acids
described herein are also contemplated herein. Such oligonucleotides are
useful as probes and primers for detecting, isolating or amplifying B.
abortus protein encoding nucleic acids.

[0060] It will be appreciated by persons skilled in the art that variants
(e.g., allelic variants) of the B. abortus protein sequences exist and
may be taken into account when designing and/or utilizing
oligonucleotides of the invention. Accordingly, it is within the scope of
the present invention to encompass such variants, with respect to the B.
abortus protein sequences disclosed herein or the oligonucleotides
targeted to specific locations on the respective genes or RNA
transcripts. Accordingly, the term "natural allelic variants" is used
herein to refer to various specific nucleotide sequences of the invention
and variants thereof that would occur in a population. The usage of
different wobble codons and genetic polymorphisms which give rise to
conservative or neutral amino acid substitutions in the encoded protein
are examples of such variants. Such variants would not demonstrate
substantially altered B. abortus protein activity or protein levels.

IV. Compositions and Methods

[0061] The present invention also encompasses compositions comprising at
least one B. abortus protein of the instant invention and at least one
pharmaceutically acceptable carrier. Such a pharmaceutical composition
may be administered, in a therapeutically effective amount (e.g., an
amount sufficient to elicit an immune response (e.g., as a vaccine)), to
a patient in need thereof. The pharmaceutical compositions of the present
invention can be administered by any suitable route, for example, by
injection (e.g., parenteral, intramuscular, intravenous, or
intraperitoneal administration), by oral, pulmonary, subcutaneous, nasal,
topical, or other modes of administration such as controlled release
devices. In general, pharmaceutical compositions and carriers of the
present invention comprise, among other things, pharmaceutically
acceptable diluents, preservatives, stabilizing agents, solubilizers,
emulsifiers, adjuvants and/or carriers. Such compositions can include
diluents of various buffer content (e.g., saline, Tris HCl, acetate,
phosphate), pH and ionic strength; and additives such as detergents and
solubilizing agents (e.g., Tween 80, Polysorbate 80), anti oxidants
(e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g.,
Thimersol, benzyl alcohol) and bulking substances (e.g., lactose,
mannitol). The compositions can be incorporated into particulate
preparations of polymeric compounds such as polylactic acid, polyglycolic
acid, etc., or into liposomes. Such compositions may influence the
physical state, stability, rate of in vivo release, and rate of in vivo
clearance of components of a pharmaceutical composition of the present
invention. Exemplary pharmaceutical compositions and carriers are
provided, e.g., in "Remington's Pharmaceutical Sciences" by E.W. Martin
(Mack Pub. Co., Easton, Pa.) and "Remington: The Science And Practice Of
Pharmacy" by Alfonso R. Gennaro (Lippincott Williams & Wilkins, 2005)
which are herein incorporated by reference. The pharmaceutical
composition of the present invention can be prepared, for example, in
liquid form, or can be in dried powder form (e.g., lyophilized).

[0062] The present invention also encompasses methods for preventing,
inhibiting, and/or treating bacterial infections, particularly B. abortus
infections. In a particular embodiment, the compositions of the instant
invention are administered as a vaccine. The pharmaceutical compositions
of the instant invention can be administered to an animal, in particular
a mammal, in order to treat/inhibit/prevent a B. abortus infection.
Examples of animals to be treated include, without limitation, domestic
livestock, cervid, bovine, elk, cattle, and bison. The pharmaceutical
compositions of the instant invention may also comprise at least one
other anti-microbial agent (e.g., antibiotic), particularly at least one
other anti-B. abortus compound/agent/vaccine. The additional anti-B.
abortus compound may also be administered in separate composition from
the B. abortus proteins of the instant invention. The compositions may be
administered at the same time or at different times (e.g., sequentially).
While the above methods describe the inhibition of a B. abortus
infection, the methods can also be employed more generally to
inhibit/treat/prevent brucellosis as well as reduce/eliminate the
symptoms associated therewith. In a particular embodiment of the instant
invention, the composition to be administered to the animal comprises at
least one, particularly at least two or three, proteins from Table 1. In
still another embodiment, the composition comprises at least one, at
least two, or all of D15, mdh, and AfuA.

[0063] The instant invention also encompasses methods for detecting a B.
abortus infection in an animal (e.g., diagnosing) and/or detecting
brucellosis in an animal. In a particular embodiment, the method
comprises 1) obtaining a biological sample from an animal and 2)
detecting the presence of at least one B. abortus protein of the instant
invention and/or an antibody specific for a B. abortus protein of the
instant invention, wherein the presence of a B. abortus protein of the
instant invention and/or antibody specific for a B. abortus protein of
the instant invention is indicative of a B. abortus infection in the
animal. While it is preferred to screen for antibodies to the proteins of
the instant invention, the instant invention also encompasses methods
wherein nucleic acid molecules which encode the B. abortus protein of the
instant invention are screened for (e.g., by using nucleic acid probes)
or the B. abortus protein of the instant invention are screened for
themselves. In a particular embodiment, the biological sample obtained
from the animal is blood or serum. In still another embodiment, the
methods comprise screening for at least one antibody specific for at
least one B. abortus protein selected from Table 1. In still another
embodiment, methods comprise screening for at least one antibody specific
for at least one, at least two, or all of D15, mdh, and AfuA
(particularly at least mdh or at least D15 and mdh).

[0064] In a particular embodiment, the methods of the instant invention
allow for the detection of a natural B. abortus infection (e.g., as
opposed to a vaccinated (e.g., B. abortus S19) host). In this embodiment,
it is preferred that the methods comprise screening for D15 and/or mdh
(e.g., antibodies specific for D15 and/or mdh).

[0065] The composition(s) of the instant invention may also be contained
within a kit. The instant invention also encompasses kits comprising a
solid support (e.g., one suitable for a lateral flow device) comprising
at least one B. abortus protein of the instant invention attached to the
surface. In a particular embodiment, the B. abortus protein(s) comprise
at least one, at least two, or all of D15, mdh, and AfuA (particularly at
least mdh or at least D15 and mdh). The kits may further comprise buffers
and detection reagents (e.g., labeled (e.g., radio-labeled or
fluorescent) secondary antibodies (e.g., specific to host antibodies))
suitable for detecting the binding of an antibody to the protein(s)
immobilized on the solid support.

[0066] The following examples describe illustrative methods of practicing
the instant invention and are not intended to limit the scope of the
invention in any way.

[0068] Serum samples from infected elk were obtained from the Wyoming Game
and Fish Department's Wildlife Disease Laboratory, a federally approved
brucellosis testing facility. Samples were evaluated using the card,
standard plate, rivanol, fluorescence polarization assay, and cELISA (to
distinguish between S19-immunity and field strain infection), and defined
as "positive" if reactions occurred in three of four tests, per federal
guidelines for Cervidae (USDA, 2003).

[0069] Five serum samples were pooled to reduce animal-to-animal
variability in reactivity to specific antigens. The sample pool was then
filter-sterilized and mixed with in vitro-grown whole cells and lysates
of B. abortus RB51, as well as E. coli BL-21[DE3] to remove in
vitro-specific antibodies, as described by others (Deb et al. (2002)
Tuberculosis 82:175-182; Chang et al. (2005) Infect. Immun.,
73:4272-4280). The adsorption process was repeated 3-5 times, and
reactivity to in vitro-grown bacteria was monitored by immunoblot to
ensure removal of antibody to constitutively expressed antigens.
Additionally, to rule out the possibility of false positives among any
reactive clones, pooled elk serum was further adsorbed with whole cells
and lysates of in vitro growth B. abortus 2308, and used to re-probe
clones (by colony lift) and/or their recombinant products (by slot blot)
reactive with the adsorbed serum used in the initial screening.

Immuno-Screening

[0070] After 5 hours of incubation at 37° C., E. coli EL-21[DE3]
colonies were lifted from the plates using Protran® nitrocellulose
membrane disks (Whatman, Kent, UK), inverted and placed on fresh LB agar
plates containing antibiotic and 1 mM IPTG. After incubation at
30° C. overnight, the membranes were exposed to chloroform vapor
for 15 minutes and allowed to dry. Once dry, the membranes were treated
with 5% skim milk powder and 0.05% Tween-20 (blotto) for 1 hour, rinsed
for 5 minutes with PBS (+0.05% Tween® 20), and incubated with
absorbed sera at a 1:500 dilution in blotto for 2 hours. After
incubation, the membranes were washed 5 times with PBS, then incubated
with a 1:5000 dilution of Alkaline Phosphatase-Conjugated, Protein G
(Rockland, Gilbertsville, Pa.) in blotto for 1 hour. Afterwards, the
membrane was washed 3 times for 5 minutes with PBS and incubated for 5
minutes with a stabilized substrate (Promega, Madison, Wis.). After color
development, membranes were rinsed with deionized water for 5 minutes.

Gene Identification

[0071] Following rinsing and drying steps, the membranes were matched to
the master plate and appropriate colonies selected and isolated. Suspect
reactive colonies were grown in broth culture and serial diluted onto new
plates. Serologic screening was repeated again and DNA extracted from
positive clones purified using the Small Plasmid Prep Kit (Qiagen,
Valencia, Calif.) and sequenced from the vector-specified T7 promoter
(University of Wyoming Nucleic Acid Exploration Facility). The sequence
was then compared against the NCBI Entrez database's B. abortus strain
9-941 and 2308 genomes for DNA/protein alignments. Subcellular
localization of putative IVIAT-identified products was determined using
the PSORTb v 2.0 algorithm (Gardy et al. (2005) Bioinformatics
21:617-623).

[0073] Sera from individual elk confirmed positive for infection with
wild-type B. abortus or immunized with B. abortus S19 were probed against
recombinant Mdh, D15, and AfuA. Purified proteins were first
electrophoresed on Tris-Tricine preparative acrylamide gels (Jule, Inc.,
Biotechnologies, Milford, Conn.), transferred via semi-dry
electroblotting to nitrocellulose membranes, and treated as similarly
described for the library immunoblotting procedure. Blot strips with 5-10
mg of immobilized protein were exposed to 1 ml of a 1:10 dilution of test
serum for 1 hour at room temperature and washed prior to addition of the
secondary antibody. Colorometric reactions were stopped after 10 minutes,
and reactivity scored as either positive or negative. Cross-reactivity of
Mdh, D15, and AfuA in B. abortus confirmed positive cattle was assessed
with bovine serum samples kindly provided by Dr. Will Reeves, ABADRL,
USDA.

Statistical Review

[0074] The two-tailed Fisher's Exact test was applied to the immunoblot
results to determine if differences observed in reactivity of any given
individual antigen between animal groups (infected, immunized, naive)
were significant. A chi-square analysis was used on the cumulative data.

Results

[0075] Brucella abortus IVI Gene Identification

[0076] The B. abortus RB51 library in E. coli, represented by a total of
approximately 35,000 clones, was probed with extensively adsorbed, pooled
immune elk serum. Ten colonies confirmed as immuno-reactive were isolated
and their Brucella-specific inserts sequenced and compared against the
genomes for B. abortus strains 9-941 and 2308 in NBCI's GenBank. All ten
loci possessed identical alleles among both strains, with the exception
of one (virJ) which was divergent in the first 85 amino acids of the
predicted product. Additionally, the products from all ten clones
remained equivalently reactive to pooled, adsorbed serum which had been
re-adsorbed with whole cells and lysates of laboratory-grown strain 2308,
suggesting that the in vitro expression profiles for the ten gene
products identified in the original screen are the same between 2308 and
its isogenic rough derivative. All but two of the predicted proteins fell
into a functional category from either the NCBI, COG (Clusters of
Orthologous Groups [of proteins]) or the cd (conserved domain) databases
(Table 1). These categories included: protein secretion, cell envelope
biogenesis, inorganic ion transport, and metabolism. Two genes, however,
predicted products of unknown function. Four of the proteins were also
determined to be unique to the Rhizobiales order (Table 1). The 10 gene
products were subsequently grouped by their predicted cellular
localization. At least seven of the antigenic proteins possess primary
and secondary structure which indicates secretion beyond the inner
membrane (Gardy et al. (2005) Bioinformatics 21:617-623).

[0077] To explore the utility of a diagnostic application for these
antigens in elk, recombinant proteins from three selected loci, D15
(1--0045), Mdh (1--1927), and AfuA (2--0539), were
purified to homogeneity, electrophoresed, electro-blotted, and probed
with serum dilutions from groups of 5-9 uninfected, vaccine-immune (S19),
and naturally infected elk. FIG. 1 shows 12 representative reactive and
non-reactive blots of the three recombinant proteins using a 1:10
dilution of individual serum samples. The serologic survey results of all
elk are summarized in Table 2. While no single antigen was uniquely
reactive between any of the animal groups, collectively, a significantly
higher frequency of reactivity in the naturally infected group was
evident (p=0.001). An analysis of individual antigens showed that
reactivity was significantly more frequent for both Mdh (p=0.007) and D15
(p=0.001) in the group of naturally infected animals, compared to
S19-immunized animals and naive animals. Although a higher frequency of
reactivity was seen with AfuA in naturally infected animals, the
difference was not statistically significant compared to S19-immunized
elk. To determine if these proteins were expressed and immunogenic in a
domestic host previously diagnosed with brucellosis, 8 confirmed
sero-positive cattle serum samples were evaluated by Western blot at the
same dilution. As shown in Table 3, the frequency of reactivity of AfuA
and Mdh was equivalent to that seen in the naturally infected elk samples
tested. Reactivity to D15, however was significantly less frequent in
sero-positive cattle compared with naturally infected elk (p=0.05).

[0078] Brucellosis caused by Brucella abortus is a significant disease in
wildlife and domestic animal populations in Wyoming and across the globe.
Infection can result in abortion, and/or persistence of the pathogen.
Recent studies show that 40% of pregnant heifers vaccinated RB51 and
challenged with fully virulent B. abortus will abort (Poester et al.
(2006) Vaccine 24:5327-5334). RB51 is also ineffective in eliciting
protective immunity to brucellosis in cervids (Olsen et al. (2006) Clin.
Vaccine Immunol., 13:1098-11103). In 2008, Wyoming suffered another
outbreak of brucellosis in a cattle herd, highlighting the state's need
for an alternative to RB51 for both cattle and cervid populations.
Application of in vivo induced antigen technology (IVIAT) to brucellosis
has facilitated the identification of numerous genes up-regulated in
vivo, whose products are immunogenic in cervids.

[0079] Furthermore, several Brucella spp. have been classed as category B
threat list agents with the potential for use as bioterrorism weapons.
Efforts to develop an effective, stable, and non-reactogenic vaccine
against brucellosis have been ongoing in several laboratories, and the
use of a live, attenuated platform has become the established benchmark
through the use of the B. abortus rough strain RB51 (Schurig et al.
(2002) Vet. Microbiol., 90:479-496). Although moderate efficacy against
Brucella-induced fetal abortions in domestic livestock (cattle) has been
reported (Elzer et al. (1998) Am. J. Vet. Res., 59:1575-1578), acceptable
levels of protection following immunization with RB51 has yet to be
demonstrated in wildlife such as elk (Cook et al. (2002) J. Wildl. Dis.,
38:18-26), and in the case of bison, results have been conflicting in
terms of the vaccine's reactogenicity (Elzer et al. (1998) J. Wildl.
Dis., 34:825-829; Olsen et al. (1999) Am. J. Vet. Res., 60:905-8; Palmer
et al. (1996) Vet. Pathol., 33:682-691). The exact nature of the
attenuation of RB51 is also unclear, although it's rough LPS phenotype is
due to at least one lesion in O-side chain biosynthesis loci (Schurig et
al. (2002) Vet. Microbiol., 90:479-496). A more systematic approach to
the induction of active protective immunity against brucellosis has been
undertaken by some laboratories through the development of subunit
vaccines (Al-Mariri et al. (2001) Infect. Immun., 69:4816-4822; He et al.
(2002) Infect. Immun., 70:2535-2543; Kaushik et al. (2010) Vet. Res.
Comm., 34:119-132; Pasquevich et al. (2009) Infect. Immun., 77:436-445;
Delpino et al. (2007) Vaccine 25:6721-6729; Cassataro et al. (2007) Clin.
Vaccine Immunol., 14:869-874). To date, the degree of success in
protecting with such vaccines depends on the ability of the candidate to
drive immunity towards a Th1-type response, emphasizing the need to
identify and characterize Brucella antigens which present T-cell epitopes
to the host (Ko et al. (2003) Clin. Microbiol. Rev., 16:65-78). Despite
the efforts to identify components for a next-generation subunit vaccine,
formulations using recombinant Brucella antigens have not been thoroughly
assessed for immunogenicity/efficacy. The discovery of additional
Brucella virulence factors thus may facilitate the development of a more
efficacious, less reactogenic, acellular product that may either be used
as a stand-alone vaccine or used to augment primary immunization with the
existing live, attenuated platform. As an example of the latter strategy,
enhanced efficacy has been reported by over-expressing Brucella
superoxide dismutase (SOD) in RB51 or complementing the strain's rough
LPS phenotype with the O-side chain biosynthesis locus, wboA (Vemulapalli
et al. (2004) Vet. Microb., 102:237-245).

[0080] As described hereinabove, the gene discovery methodology, known as
in vivo-induced antigen technology (IVIAT), has been applied to identify
B. abortus virulence genes up-regulated during infection in elk (Cervis
elaphus), and as a result ten loci with gene products potentially
important to survival of the pathogen in this host have been identified.
Furthermore, the conserved nature of most of these gene products has led
to the conclusion that they also may be requisite virulence effectors in
other Brucella susceptible hosts. As a preliminary approach to confirming
this hypothesis, five of these in vivo-induced (IVI) products have been
selected for further characterization in a surrogate murine model for B.
abortus colonization: a conserved outer membrane protein, D15; a
gluconeogenic enzyme, malate dehydrogenase (Mdh); a periplasmic component
of an ABC transport system, AfuA; a component of the Type-IV secretion
system (TOSS) VirJ; and a lipoprotein of unknown function BAB1--0187
(referred to as 0187). Three additional conserved genes based on high
amino acid sequence similarity with loci identified through Yersinia
pestis IVIAT and previous reports of a role in Brucella pathogenesis
(Andrews et al. (2010) Vector-Borne and Zoonotic Dis., 10(8):749-756;
Spera et al. (2006) Proc. Nat. Acad. Sci., 103:16514-16519) were also
targeted: a proline epimerase (PrpA; BAB1--1800 (strain 2308)), an
auto-secreting (Type-V) surface antigen (Hia; BruAb1--0072 (9-941)),
the other encoding a soluble lytic transglycosylase (MltE;
BruAb1--0661 (9-941)).

Materials and Methods

Bacterial Strains and Growth Conditions

[0081] Brucella abortus S19, was kindly provided by the Colorado Serum
Company (Denver, Colo.), and was used exclusively for this study in the
mouse colonization/infection model. Brain-heart infusion broth cultures
were typically grown overnight at 37° C., serially diluted after
three washes in sterile PBS, followed by plating to determine a viable
cell count correlate with optical density at 600 nm.

In Vivo Gene Expression, RNA Extraction, and RT-PCR

[0082] Ten BALB/c mice were infected with 1×107 cfu of B.
abortus S19 i.p. Mice were splenectomized and tissues stored in
RNAlater® (Ambion, Austin, Tex.). Tissues were homogenized and RNA
isolated with the RiboPure®-Bacteria Kit (Ambion, Austin, Tex.).
Isolated RNA was transcribed to cDNA using RETROscript® (Ambion,
Austin, Tex.) and cDNA targets amplified by PfuTurbo® DNA Polymerase
(Stratagene, La Jolla, Calif.) in a one-step reaction. Amplification of a
segment of the 16S subunit of B. Abortus S19 was used as a positive
control; negative controls were included for each gene and contained all
the reaction components except reverse transcriptase. In addition a
negative control was employed which lacked RNA template to confirm the
absence of DNA contamination in the reaction. Concentration of PCR
product in gel bands was assessed using Quantity One 4.6 (Bio-rad,
Hercules, Calif.).

[0085] Purified proteins were mixed with a 1:7 dilution of aluminum
hydroxide adjuvant (Alhydrogel; Superfos, Denmark) in PBS and adsorbed
overnight at 4° C. at a concentration of 150 μg/mL.

Animal Studies

[0086] All animals utilized in this study were cared for according to
strict adherence to the Policies and Regulations established by the US
Public Health Service "Humane Care and Use of Laboratory Animals" and an
approved animal protocol from the University of Wyoming Institutional
Animal Care and Use Committee (IACUC) (DHHS Assurance #A3216-1). Animals
were euthanized by the AVMA approved method of cervical dislocation.

[0087] Ten to 30 six-week old, female BALB/c mice received 30 μg of
recombinant protein subcutaneously in 200 μL of adjuvant at one site.
Additional control mice were treated with adjuvant only in the same
manner. Immunization regimen consisted of a prime and two boosts, 21 days
apart. Retro-orbital bleeds were performed to assess antibody titers by
Western blot. Animals were challenged with 5×104 organisms of
B. abortus S19 occurred at 14 days after the second boost. Five mice from
each group were sacrificed at specific time points. Spleens were removed,
weighed, homogenized, used to determine whole organ bacterial load
following serial dilution of the homogenates in 1× Sterile PBS and
plating on blood agar. The remaining homogenates were stored at
-40° C. for cytokine analysis.

Cytokine Analysis

[0088] Supernatants from spleen homogenates were used in QuantiKine®
ELISA Assays (R&D Systems, Minneapolis, Minn.) to quantify IL-12p70,
IL-4, and IFN-γ cytokine levels in the spleen.

Statistics

[0089] All statistical analysis was completed in the software package SAS
9.1 Enterprise (SAS Corporation, Cary, N.C.). ANOVA was used to compare
means of groups and Least Significant Difference (LSD) was used to
determine mean separations between the groups. α=0.05; p values are
listed in text.

Results

S19 Infection Kinetics

[0090] To establish the colonization kinetics of B. abortus S19 in BALB/c
mice, thirty naive animals were infected with S19 at 5×104 CFU
and five animals sacrificed at 7, 14, 21, 28, 42, and 70 days
post-infection. As shown in FIG. 5, bacterial loads in spleens peaked in
two weeks at 8×107 CFU before gradually declining to
6×103 CFU in 6 weeks. These observations were consistent with
a previous report of S19 colonization kinetics in mice (Montaraz et al.
(1986) Infect. Immun., 53:245-251), however in the instant study, at 10
weeks post infection, organisms were still able to be cultured from
spleens in 60% of the animals. Also, S19-induced splenomegaly in mice
correlated with the bacterial load at specific time points, peaking
between 14 and 21 days post-infection, and declining by day 28 (Table 4).

[0091] As seen in FIG. 6, the vaccination with recombinant Mdh reduced B.
abortus S19 colonization in the spleen of BALB/c mice. 15 mice were
immunized with mdh. 5 mice were sacrificed at 7, 14, and 21 days
post-infection and bacterial loads in the spleen were determined.
Alhydrogel® only animals represent pooled data from all vaccination
studies.

[0092] In another experiment, thirty mice were vaccinated with purified
Mdh, MltE, or adjuvant-only. After hyper-immunization with the
recombinant proteins, the sera was assessed for antibody. End-point
titers from mice immunized with the recombinant protein ranged from a
1:1000 to 1:5000. Serum from mice receiving alhydrogel alone was
non-reactive against any of the proteins. After challenge, bacterial
loads in the spleens were determined at 7, 14, 21, and 28 days
post-infection. As shown in Table 5, mice vaccinated with Mdh showed a
markedly significant decrease in bacterial colonization at 14 dpi
providing 2.75 log units of clearance and 2 log units of clearance at 21
days post-infection compared to adjuvant-only treated animals
(p<0.001). Bacterial loads measured in MltE-immunized mice were no
different than the adjuvant-only controls. Mdh-immunized animals also
displayed extended splenomegaly which remained elevated relative to the
adjuvant-only animals at 28 days post-infection (Table 5). By 42 days,
the Mdh-immunized animals had completely cleared the infection, while S19
was still cultured from spleens of the adjuvant-only mice at the same
time point.

[0093] In another experiment, fifteen mice each received alhydrogel only,
AfuA, Hia or D15 recombinant proteins adsorbed to the adjuvant. Mice were
primed and boosted twice, and serum was collected to assay for antibody
titers. Reactivity of the immune sera to all three antigens was
comparable to that of Mdh immunized mice. The animals were challenged as
before and bacterial loads in the spleens assessed at 14, 21, and 28 days
post-infection. As shown in Table 6, in contrast to Mdh, there was no
significant difference at two weeks between the adjuvant-only and
antigen-immunized animal groups, however at three weeks post-infection
all three experimental groups differed significantly from the adjuvant
only group with D15 providing the most pronounced effect of 1.41 log
units of clearance (p<0.001). As observed with Mdh-immunized animals,
splenomegaly remained consistently elevated in all test groups at 28 days
post-infection relative to the adjuvant-only control animals (Table 7).

[0094] Another iteration was then conducted to evaluate VirJ, 0187, and
PrpA. Following the same methods as above, antibody titers were
determined to be >1:5000 (VirJ, PrpA and 0187). Mice were challenged
with S19 and splenic colony counts were performed as described previously
at 14 and 21 days post-infection. No significant difference was found
between adjuvant-only animals and those receiving any of the three
antigens (Table 8), although all immune animals displayed some degree of
splenomegaly (Table 7).

[0095] Levels of IL-12p70, IL-4 and IFN-γ in the splenic homogenates
were quantified from the five selected animal groups immunized with
antigens which had an effect on bacterial load and/or clearance rate. No
detectable IL-12p70 or IL-4 groups immunized with AfuA, Hia, D15, or
adjuvant alone was observed. IL-4 was, however, detected in
Mdh-vaccinated mice, although at low levels.

[0096] IFN-γ was next assessed in vaccinated mice after challenge. 5
spleen tissue homogenates from each group and timepoint were assayed by
ELISA. As shown in FIG. 7, at all sampling times, mice vaccinated with
Mdh showed significantly higher levels of this cytokine (p<0.05),
compared to the mice receiving alhydrogel alone (or AfuA or D15). By 21
and 28 days post-infection, IFN-γ levels among all groups had
declined with the exception of the Mdh immune group, in which IFN-γ
remained significantly elevated (p<0.05; FIG. 7). Additionally, BALB/c
mice vaccinated with Mdh show increased splenomegaly. Indeed, at one
week, Mdh vaccinated spleens were 717.6±209.2 mg whereas as MltE
spleens were 460.6±181.0 mg and Alhydrogel® mice were
506.8±160.4 mg. At two weeks, Mdh vaccinated spleens were
1057.8±43.5 mg whereas as MltE spleens were 986.2±102.9 mg and
Alhydrogel® mice were 878.2±52.5 mg.

[0097] As seen in FIG. 8, immunization with recombinant proteins promotes
faster clearance of B. abortus S19 at three weeks post infection in
BALB/c mice.

[0099] In addition to the above, vaccination studies in mice with D15 and
AfuA have shown that each of these proteins is capable of eliciting an
immune response in mice challenged with B. abortus S19.

In Vivo Assessment of IVI Gene Up-Regulation During S19 Infection in Mice

[0100] "Short-unique" regions of selected IVI gene targets identified by
IVIAT from elk infected with wild-type B. abortus were selected for
construction of RT-PCR primers. Ten BALB/c mice were subsequently
infected with S19, five of which were splenectomised at 24 and 48 hours
post-infection, and bacterial mRNA isolated. Additionally, S19 was grown
to mid-log phase in vitro and mRNA extracted for comparison. Analysis
showed up-regulation during both 24 and 48 hours post-infection of afuA,
mdh, and 0187 (FIG. 10). In contrast, D15 mRNA was not detected in either
the in vitro or in vivo samples, even after performing several different
nested RT-PCR reactions. Hia transcript was, as expected, expressed at
equivalent levels in vitro and during infection.

[0102] Vaccination with purified B. abortus Mdh resulted in significantly
reduced colonization and more rapid clearance of S19 in the BALB/c mouse.
Interestingly, Mdh was the only recombinant protein of the five antigens
examined which facilitated some level of clearance that elicited a
significant IFN-γ response, a cytokine critical for the activation
of macrophages and a requisite for controlling Brucella infections
(Baldwin et al. (2006) Crit. Rev. Immunol., 26:407-442). It is therefore
likely that prolonged elevated levels of IFN-γ in mice vaccinated
with Mdh contribute to the reduction in the colonization by S19 in these
immune animals. The nature of Mdh-induced immune-mediated enhanced
clearance is unclear at present, however, it is possible that an
auxiliary virulence function of the enzyme may be neutralized by a robust
immune response directed toward it. The presence of IL-4 and absence of
IL-12 also suggest that mice vaccinated with Mdh induce a Th2-biased
response leading to clearance immunity mediated by antibody. In fact, a
search for putative T- and B-cell epitopes across the amino acid sequence
of the protein revealed only the latter. This hypothesis may seem
contrary to the traditional notion that only a Th1-biased response can
reduce intracellular bacterial load in the Brucella-infected host.
Indeed, previous experiments with other facultative intracellular
pathogens such as Yersinia pestis have demonstrated that antibody alone
can confer protection against challenge (Sofer-Podesta et al. (2009)
Infect. Immun., 77:1561-1568). In the case of B. abortus, an "optimized"
Th2 response might also contribute significantly to clearing infection.

[0103] Although, immunity to AfuA and D15 failed to elicit more rapid
clearance of S19, bacterial loads were significantly reduced in animals
immunized with either of these two proteins. In contrast to AfuA and Mdh,
D15 expression was not evident early in S19 colonization, thus D15 may be
relevant at a later stage of infection in the mouse. Curiously, as
described hereinabove, an antibody to Mdh and D15 was not detected in
S19-immunized elk, indicating that both proteins may be regulated
differently in cervids.

[0104] Hia was not identified as an IVI gene initially, and consistent
with this finding, was subsequently was found to be constitutively
expressed based on an mRNA analysis. The selection of this gene product
was based on similar homology to Y. pestis protein and previous reports
in the literature of involvement as a virulence factor (Alamuri Pet al.
(2010) Infect. Immun., 78:4882-4894). This type-V auto-secreted antigen,
induced a greater level of extended splenomegaly compared to the other
recombinant proteins, including Mdh. However, the increased inflammation
did not correlate with heightened production of IFN-γ. This
observation indicates that immunity to Hia may increase the inflammatory
response during infection by mechanisms not related to IFN-γ, such
as TNF-α or IL-1. Although immunization with Hia reduced bacterial
load in the mouse model comparable to D15 and AfuA, it failed to induce
more rapid clearance.

[0105] As with MltE, 0187, PrpA, and VirJ failed to reduce bacterial load
and/or alter clearance kinetics compared to adjuvant-only controls. 0187
is a putative lipoprotein that shares significant homology with the well
characterized BA14K protein. Previous studies have demonstrated that
BA14K was able to induce a Th1 response and induce IL-12 secretion
(Chirhart-Gilleland et al. (1998) Infect. Immun., 66:4000-4003). 0187 was
not expressed from B. abortus as a full length protein but it was stably
expressed as a truncated form shortened by 27 amino acids from the
N-terminus. It is possible that this truncation could have resulted in
conformation changes leading to an inability to induce clearance
immunity.

[0106] PrpA, which encodes a proline epimerase was described as a B-cell
polyclonal activator and inducer of IL-10 (Spera et al. (2006) Proc. Nat.
Acad. Sci., 103:16514-16519), suggested that immunity to this protein may
promote clearance in the model based on the pivotal role IL-10 plays in
Brucella pathogenesis (Baldwin et al. (2006) Crit. Rev. Immunol.,
26:407-442; Fernandes et al. (1995) Infect. Immun., 63:1130-1133). Mice
immunized with PrpA during early infection actually had splenic counts
higher than animals sham-immunized with adjuvant alone. It is possible
that a secondary exposure to PrpA results in host immune dysregulation
early during the course of infection.

[0107] The data above strongly suggested that the Type-IV secretion system
(T4SS) accessory protein, VirJ, is up-regulated during wild type B.
abortus infection in elk. Animals immunized with S19 however did not
generate a humoral response to the protein, which suggests a difference
in the way this secreton is utilized by S19 in cervids. Consistent with
this finding, in the S19 murine colonization model, VirJ appears to be
slightly down-regulated at least during early stages of infection. A
BLAST analysis upstream and downstream of VirJ revealed no differences
between S19, 2308 or 9-941 (Wyoming strain) genome sequences, suggesting
the involvement of a distal regulatory element(s) in controlling
expression in S19. The function of VirJ, as assessed in other pathogens,
is suspected to be as a periplasmic chaperone responsible for assisting
substrates in associating with a "pusher" pilus before translocation
through the T4SS, typically thought to be required for full virulence in
this pathogen (Zhong et al. (2009) Microbiol., 155:3392-3402). In this
regard, VirJ may be studied in a B. abortus 2308 challenge model.

[0108] Data from the model system is in agreement with that previously
published for S19 colonization in BALB/c mice, and also demonstrated for
the first time that mice remain colonized with S19 at ten weeks post
infection. The vaccination efforts with a single recombinant protein,
Mdh, coincide with previously reported data on mice vaccinated with RB51
in terms of the subsequent cytokine responses post-challenge (Wang et al.
(2010) FEMS Microbiol. Letters, 303:92-100). IFN-γ levels peak
between 6 and 7 days then begin to slowly decline, albeit remaining
sustained for weeks (Wang et al. (2010) FEMS Microbiol. Letters,
303:92-100). RB51 vaccinates also lack significant production IL-12p70 or
high levels of IL-4 upon challenge (Wang et al. (2010) FEMS Microbiol.
Letters, 303:92-100). Notably, BALB/c mice tend to be more biased towards
humoral responses (Schurig et al. (2002) Vet. Microbiol., 90:479-496;
Baldwin et al. (2006) Crit. Rev. Immunol., 26:407-442; Wang et al. (2010)
FEMS Microbiol. Letters, 303:92-100). As predicted, the S19 data shows
that a pro-inflammatory response is suppressed in naive animals and
behaves similarly to strain 2308 in this respect (Baldwin et al. (2006)
Crit. Rev. Immunol., 26:407-442). This indicates that a shift in cytokine
production levels is important in providing a more efficacious immune
response to brucellosis.

[0109] Taken together, these data indicate the potential for use of the
gluconeogenic enzyme, malate dehydrogenase, as a recombinant subunit
vaccine candidate for brucellosis. AfuA and D15 also represent subunit
vaccine candidates, particularly when used together and/or in combination
with Mdh. Collectively, the in vivo data gathered from the S19 murine
colonization model indicate that vaccination with at least three of the
IVIAT antigens conferred an enhanced ability of the host to respond to
infection, establishing the utility of this methodology for the
identification of potential vaccine candidates against brucellosis.

[0110] A number of publications and patent documents are cited throughout
the foregoing specification in order to describe the state of the art to
which this invention pertains. The entire disclosure of each of these
citations is incorporated by reference herein.

[0111] While certain of the preferred embodiments of the present invention
have been described and specifically exemplified above, it is not
intended that the invention be limited to such embodiments. Various
modifications may be made thereto without departing from the scope and
spirit of the present invention, as set forth in the following claims.